Our general objective is to elucidate the structure and function of heterochromatin. Also, we are interested in the mechanics of sequence divergence of highly repetitive DNA during evolution. We chose the large heterochromatic chromosome of Drosophila nasutoides as a model system for these studies, mainly due to our finding that this chromosome contains almost nothing but satellite DNA components which amount to about 50% of the total genome. For this study, we set up the following specific goals in the past year. Preliminary results are described below: (a) Preparation of metaphase chromosomes using permeable embryos. The ultimate purpose of this project is to isolate the large heterochromatic chromosome so that we can do biochemical experiments with this material. Our pilot experiments showed that about 70% of the permeable embryos are at metaphase. Scaled-up experiments are in progress. (b) Isolation and characterization of chromosomal protein(s) which binds preferentially to satellite DNAs, in order to understand the condensation of heterochromatin. We have not been successful yet. Our search continues. (c) DNAase susceptibility of various chromatins containing main band and satellite DNAs in order to understand the architecture of chromatin inside the nucleus. Isolated nuclei were treated with DNAaseI and Staph nuclease. DNAs were purified and displayed on agarose gel. Southern blot hybridizations using various DNA probes are in progress. (d) Restriction analysis of satellite II DNA which is very highly diverged. Limit digests of this DNA by EcoRI, Sal I or AluI reveal multimeric fragmentation patterns with the size of the monomer being 96 base pairs. A multimeric pattern was expected for a diverged DNA, but we did not expect that all three enzymes produce identical patterns. Our double-digestion experiments and restriction analysis of cloned satellite II fragments show that; (1) there is a sequence in this DNA containing almost exclusively of EcoRI site, (2) there is a sequence containing primarily of SalI site and some of AluI site, and (3) there is a sequence containing primarily of AluI site and some of SalI site. The latter point is not firm yet.